Cell adhesion is a process by which cells associate with each other and migrate towards a specific target localized within the extracellular matrix. Specialized molecules, called cell adhesion molecules (CAMs), mediate these reactions. CAMs have been demonstrated to participate in various cell-cell, cell-extracellular matrix, and platelet-platelet interactions. CAMs influence the leukocytes' adhesion to the vascular endothelium, their transendothelial migration, retention at extravascular sites, and activation of T cells and eosinophils. These processes are central to the pathogenesis of inflammatory and autoimmune diseases. Therefore, CAMs are considered potential targets for treating such disorders.
CAMs can be classified into three groups: integrins, selectins, and the immunoglobulin superfamily. Of these, integrins are the key mediators in the adhesive interactions between hemopoietic cells and their microenvironment. They are comprised of alpha-beta heterodimers and integrate signals from the outside to the inside of cells, and vice versa. Integrins can be classified on the basis of the beta subunits they contain. For example, the beta-1 subfamily contains beta-1 subunit noncovalently linked to one of the 10 different alpha subunits.
The alpha-4 beta-1 integrin, also known as VLA4 (very late activation antigen 4), is a member of the beta-1 integrin family and comprises alpha-4 and beta-1 subunits. VLA4 interacts with two specific ligands—the vascular cell adhesion molecule (VCAM-1) and the CS1 region of the protein fibronectin. Adhesion mediated by VLA4 is central to the process of transendothelial migration of leukocytes. Ligation of VLA4 is followed by gross rearrangement of the cytoskeleton, leading to flattening of cells along the blood vessel wall, followed by expression of specific molecules that digest the endothelial cell wall and diapedesis. Once in the extraluminal region, the interactions of VLA4 with extracellular fibronectin play a crucial role in the migration of leukocytes to the site of inflammation, T cell proliferation, expression of cytokines and inflammatory mediators. Additionally, VLA4 ligation provides co-stimulatory signals to the leukocytes, resulting in enhanced immunoreactivity. Thus, appropriate VLA4 antagonists would, in theory, ameliorate the immune response through a twofold action—inhibition of T cell recruitment at the site of inflammation and inhibition of co-stimulatory activation of immune cells.
In this respect, inhibitors of VLA4 interactions have been demonstrated to exhibit beneficial therapeutic effects in several animal models of inflammatory and allergic diseases, including sheep allergic asthma (Abraham et al, J. Clin. Invest. 1994; 93:776); arthritis (Wahl et al, J. Clin. Invest. 1994; 94:655); experimental allergic encephalomyelitis (Yednock et al, Nature (Lond), 1992; 356:63 and Baron et al, J. Exp. Med. 1993; 177:57); contact hypersensitivity (Chisolm et al, Eur J. Immunol. 1993; 23:682); type I diabetes (Yang et al, Proc. Natl. Acad. Sci. (USA) 1993; 90:10494); and inflammatory bowel disease (Podolsky et al, J. Clin. Invest. 1993; 92:372).
The CS1 moiety region of fibronectin involved in the interaction with VLA4 was identified as the tripeptide Leu-Asp-Val (LDV) (Komoriya et al, J. Biol. Chem. 1991; 266:15075). Several peptides containing the LDV sequence were synthesized and shown to inhibit the in vivo interaction of VLA4 to its ligands (Ferguson et al, Proc. Natl. Acad. Sci. (USA) 1991; 88:8072; Wahl et al, J. Clin. Invest. 1994; 94:655; Nowlin et al, J. Biol. Chem. 1993; 268(27):20352; and PCT publication WO91/4862).
Despite these advances a need for small and specific inhibitors of VLA4-dependent cell adhesion molecules remains. Ideally, such inhibitors are water soluble with oral efficacy. Such compounds would provide useful agents for the treatment, prevention or suppression of various inflammatory pathologies mediated by VLA4 binding.
It is generally known that isopropylidene and benzylidene groups are the most commonly used protective groups in carbohydrate chemistry. Although both groups are introduced into a molecule under similar conditions, the location of the protection can be quite different, and this difference is directly related to the stability of each protected molecule. Since protection normally occurs under conditions that allow reversibility, the reaction proceeds until equilibrium is reached. The distribution of products at equilibrium is determined by their relative thermodynamic stabilities. In other words, these reactions are thermodynamically controlled. Benzylidene groups prefer to be part of 6-membered ring acetals, while the ketals resulting from acetonation generally are 5-membered rings. The difference is attributed to the effect of the methyl and phenyl substituents on the stability of the particular ring systems. These blocking methods are described in the U.S. Pat. Nos. 2,715,121, 4,056,322, 4,735,934, 4,996,195 and 5,010,058, the disclosures of which are incorporated herein by reference. Other blocking methods are also described in J. Carbohydr. Chem. 1985; 4:227 and 1984; 3:331; Methods in Carbohydr. Chem. 1962; 1:107 and 1962; 1:191; Can J. Chem. 1984; 62:2728, 1969; 47:1195, 1455, and 1970; 48:1754, all incorporated herein by reference. The prior art reveals that D-glucose is blocked at the 1,2; 5,6-positions with either the isopropylidene or cyclohexylidene blocking group, leaving the 3-position open to undergo derivatization. The therapeutic activity of hexoses and their derivatives are also disclosed in some of the above-cited prior art.
The compounds of the present invention were screened for inhibitory activity in VLA4-mediated cell adhesion assay and the classical murine hypersensitivity assay in mice. Several compounds exhibited significant inhibitory activity in both tests. The salts of these compounds could be easily solubilized in water and used in the treatment of chronic, cell adhesion-mediated, allergic, autoimmune and inflammatory disorders, such as bronchial asthma and rheumatoid arthritis. Some of the prior art describes development of peptide derivatives as cell adhesion antagonists for treatment of these diseases. However, because treatment of chronic diseases requires prolonged (mid-term to long-term) administration of drugs, the development of specific, orally available cell adhesion inhibitors would be very beneficial.
There is no example available in the prior art, wherein the compounds, containing a sugar nucleus coupled with carbamate moiety, of the present invention are used as therapy for the inhibition, prevention and suppression of VLA4-mediated cell adhesion and pathologies associated with that adhesion.